Adenovirus serotype 5 neutralizing antibodies target both hexon and fiber following vaccination and natural infection.

The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). However, the role of NAbs directed against other capsid components, particularly the adenovirus fiber, remains unclear. Here we show that Ad5 NAbs target both hexon and fiber following vaccination and natural infection. Utilizing neutralization assays with capsid chimeric vectors, we observed that NAb responses to hexon appeared dominant and NAb responses against fiber were subdominant in sera from vaccinated mice, vaccinated humans, and naturally exposed humans. A novel chimeric Ad5 vector in which both the hexon HVRs and the fiber knob were exchanged nearly completely evaded Ad5-specific NAbs both in vitro and in vivo.

Adenovirus serotype 5-specific neutralizing antibodies target multiple hexon hypervariable regions.

The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). We previously reported that replacing all seven HVRs with those from the rare serotype virus Ad48 resulted in a chimeric Ad5HVR48(1-7) vector that largely evaded preexisting Ad5 immunity in mice and rhesus monkeys. In this study, we evaluated the extent to which Ad5-specific NAbs are directed against various HVRs. We constructed partial HVR-chimeric Ad5 vectors with only a subset of HVRs exchanged, and we utilized these vectors in both NAb assays and murine immunogenicity studies with and without baseline Ad5 immunity. Our results demonstrate that Ad5-specific NAbs target multiple HVRs, suggesting that replacing all HVRs is required to optimize evasion of anti-Ad5 immunity. These data have important implications for the development of novel vectors for both vaccines and gene therapy.

Breadth of neutralizing antibodies elicited by stable, homogeneous clade A and clade C HIV-1 gp140 envelope trimers in guinea pigs.

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.

HIV-1 gp120-induced migration of dendritic cells is regulated by a novel kinase cascade involving Pyk2, p38 MAP kinase, and LSP1.

Targeting dendritic cell (DC) functions such as migration is a pivotal mechanism used by HIV-1 to disseminate within the host. The HIV-1 envelope protein is the most important of the virally encoded proteins that exploits the migratory capacity of DCs. In the present study, we elucidated the signaling machinery involved in migration of immature DCs (iDCs) in response to HIV-1 envelope protein. We observed that M-tropic HIV-1 glycoprotein 120 (gp120) induces phosphorylation of the nonreceptor tyrosine kinase, Pyk2. Inhibition of Pyk2 activity using a pharmacologic inhibitor, kinase-inactive Pyk2 mutant, and Pyk2-specific small interfering RNA blocked gp120-induced chemotaxis, confirming the role of Pyk2 in iDC migration. In addition, we also illustrated the importance of Pyk2 in iDC migration induced by virion-associated envelope protein, using aldithriol-2-inactivated M-tropic HIV-1 virus. Further analysis of the downstream signaling mechanisms involved in gp120-induced migration revealed that Pyk2 activates p38 mitogen-activated protein kinase, which in turn activates the F-actin-binding protein, leukocyte-specific protein 1, and enhances its association with actin. Taken together, our studies provide an insight into a novel gp120-mediated pathway that regulates DC chemotaxis and contributes to the dissemination of HIV-1 within an infected person.

LPS-induced MCP-1 expression in human microvascular endothelial cells is mediated by the tyrosine kinase, Pyk2 via the p38 MAPK/NF-kappaB-dependent pathway.

Bacterial endotoxin (lipopolysaccharide or LPS) has potent pro-inflammatory properties and acts on many cell types including endothelial cells. Secretion of the CC chemokine, MCP-1 (CCL2) by LPS-activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in LPS-induced MCP-1 production in endothelial cells is not well understood. Using human microvascular endothelial cells (HMVEC), we analyzed the involvement of the non-receptor tyrosine kinase, Pyk2, in LPS-mediated MCP-1 production. There was a marked activation of the non-receptor tyrosine kinase, Pyk2, in response to LPS. Inhibition of Pyk2 activity using a pharmacological inhibitor, Tyrphostin A9 significantly attenuated LPS-induced Pyk2 tyrosine phosphorylation, p38 MAP kinase (MAPK) activation, NF-kappaB activation, and MCP-1 expression. Furthermore, specific inactivation of Pyk2 activity by transducing microvascular endothelial cells with catalytically inactive Pyk2 mutant (AAV-Pyk2MT) or Pyk2-specific siRNA significantly blocked LPS-induced MCP-1 production. The supernatants of these LPS-stimulated cells with attenuated Pyk2 activity demonstrated decreased trans-endothelial monocyte migration in comparison to LPS-treated controls, thus confirming the inhibition of functional MCP-1 production. In summary, our data suggest a critical role for the Pyk2 mediated pathway involving p38 MAP kinase and NF-kappaB in LPS-induced MCP-1 production in human microvascular endothelial cells.

Vaccinia virus K1L protein mediates host-range function in RK-13 cells via ankyrin repeat and may interact with a cellular GTPase-activating protein.

The K1L protein of vaccinia virus is required for its growth in certain cell lines (RK-13 and human). The cowpox host-range protein CP77 has been shown to complement K1L function in RK-13 cells, despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats of K1L protein in RK-13 cells. The growth of a recombinant vaccinia virus, with K1L gene mutated in the most conserved ankyrin repeat, was severely impaired. Infection with the mutant virus caused shutdown of cellular and viral protein synthesis early in infection. We also investigated the interaction of K1L protein with cellular proteins and found that K1L interacts with the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein, which may be interacting with K1L.